Accurate mass determination by multiple sprayers nano-electrospray mass spectrometry on a magnetic sector instrument

A new technique for accurate mass determination by using multiple sprayers nano-electrospray ionization mass spectrometry (nano-ESI-MS) on a magnetic sector instrument is described. Metal coated glass capillaries were used as nano-ESI sprayers. One of the sprayers was used for the reference compound solution, and others were used for the introduction of sample solutions. The spectra of the different compounds were obtained by shifting each sprayer's position relative to the sampling orifice. The accurate masses of several standard compounds were obtained with good accuracy, without problems arising from differences in ionization efficiency between the sample compounds and reference compound.     

Electron-capture negative ionization calibrants for magnetic sector mass spectrometers

Fomblin [poly(perfluoropropylene oxide)], PFK (perfluorokerosene) and FC-43 (perfluorotributylamine) are investigated as mass calibrants in electron-capture negative ionization mass spectrometry on a magnetic sector hybrod mass spectrometer. This work provides exact negative ion mass tables of these three calibrants obtained using high-resolution peak matching of representative ions of the calibrants against ions of known mass. The suitability of these calibrants for full data system control in the negative ionization mode is demonstrated for the techniques of high-resolution scanning, selected ion recording and calibration of the quadrupole sector of a hybrid instrument.     

Characterization of complex assemblages of organic acids in geological samples by negative electrospray ionization mass spectrometry using a double‐focusing magnetic sector field mass spectrometer

Four different geological sample types (a crude oil, a crude oil asphaltene, a reservoir core extract and a reservoir core asphaltene) have been characterized by negative ionization electrospray mass spectrometry at low and high mass resolution using a double‐focusing magnetic sector field mass spectrometer. The mass range, shape of the spectra and the signal distribution of the acidic constituents as well as the average molecular weights, the total ion abundance and signal intensity in the spectra were compared for the different sample types. Nominal mass classes have been evaluated and Kendrick mass plots were generated in order to identify homologous series. For the crude oil sample, accurate mass assignments were made by high‐resolution double‐focusing magnetic sector field mass spectrometry (DFMSFMS) and were compared with those obtained by negative ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). With both instrument types, compounds with the molecular composition C_nH_2n+zO₂, among which carboxylic acids predominated, were the main acidic compound class detectable in negative ESI mass spectra. Good agreement was achieved for the double bond class distribution and the carbon number distribution of the O₂ class. In addition, minor compound classes could be identified using FTICRMS. Copyright © 2010 John Wiley & Sons, Ltd.     

A comparison of accurate mass techniques for the structural elucidation of fluconazole

Mass spectrometry plays a major role in the structural elucidation and characterisation of drug candidates and related substances. Accurate mass data allow the mathematical prediction of molecular formula of both precursor and fragment ions. In this paper, a comparison of the accurate mass data obtained for the fragmentation of fluconazole, an antifungal drug, by three different methods is made: electron ionisation (EI) using a magnetic sector instrument; electrospray ionisation (ES) using a Fourier transform ion cyclotron mass spectrometer (FTICRMS); and ES using a quadrupole-time-of-flight mass spectrometer (Q-ToF). It is clear from the data obtained that mass accuracy is not simply a function of instrument resolution. The subtle differences observed between collisionally activated dissociation (CAD) and sustained off-resonance collisionally activated dissociation (SORI-CAD) spectra are explained as a consequence of the excitation process. The advantages and disadvantages of the three techniques are discussed within the context of structural elucidation.     

Comparative performance study of ICP mass spectrometers by means of U "isotopic measurements"

The performance of four commercially available ICPMS instruments of three different types was compared by means of uranium "isotopic measurements". Examined were two quadrupole sector (different generation, different manufacturer), one single detector double focusing magnetic sector and one multiple collector double focusing magnetic sector instruments. The same samples of the IRMM-072 series were used under routine conditions to measure the 233U/235U and the 233U/238U ratios which, in these samples, vary over almost three orders of magnitude from approximately 1 to approximately 2 x 10(-3). Within expanded (k = 2) uncertainties, good agreement was observed between the certified values and the data internally corrected for mass-discrimination effects. The magnitude of the evaluated uncertainties was different for each type of instrument. With the multiple collector instrument, expanded uncertainties varied from +/- 0.04% to +/- 0.24% for the 233U/235U ratio, and from +/- 0.08% to +/- 0.27% for the 233U/238U ratio. They were approximately 1 to 5 times larger with the single detector magnetic sector instrument, and approximately 10 to 25 times larger with both quadrupole sector instruments. With the multiple collector instrument, repeatability of the measurements seemed to be limited by the difficulty of correcting properly for instrumental background, whereas with the single detector magnetic sector instrument the counting statistics was the only limitation (on smallest ratios). Apparent mass-discrimination was clearly found to be larger but more reproducible (and hence easier to correct for) in the case of magnetic sector instruments than for both quadrupole sector instruments. If space charge effects were the main source of mass-discrimination for all instruments, these results are in contradiction with the hypothesis of the size of mass-discrimination decreasing with the acceleration voltage. With the single detector magnetic sector instrument in particular (when operated by changing the ion energy only), our results pointed at more than only one major source of mass-discrimination, with variable size depending on the ratios measured.     

A new linked scan for reversed geometry mass spectrometers

A new linked scan is described by which fragmentations occurring in the second field free region of a two sector instrument can be monitored. The scan can be used only if the first stage allows selection of ions according to their masses. The magnetic sector and electric sector are scanned in unison so that the product B²E is maintained constant. A spectrum of all parents of a preselected daughter ion is obtained and the resolution depends only on the mass resolution of the magnetic sector.     

Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection

A method using capillary electrophoresis-mass spectrometry (CE-MS) was developed for the structural elucidation of bupivacaine and metabolites in rat urine. Prior to CE-MS analysis, solid-phase extraction (SPE) was used for sample cleanup and preconcentration purposes. Exact mass and tandem mass spectrometric (MS/MS) experiments were performed to obtain structural information about the unknown metabolites. Two instruments with different mass analyzers were used for mass spectrometric detection. A quadrupole time-of-flight (Q-TOF) and a magnetic sector hybrid instrument were coupled to CE and used for the analysis of urine extracts. Hydroxybupivacaine as well as five other isomerically different metabolites were detected including methoxylated bupivacaine.     

The observation of broad metastable ion peaks in the surface-induced dissociation mass spectra of protonated and cationated peptides

The product ion mass spectra of protonated and cationated peptides of relative molecular mass (RMM) 555–574 Da have been obtained by surface-induced dissociation of MH⁺ and [M + Cat]⁻ ions in a four-sector tandem mass spectrometer equipped with a specially designed collision cell. A linked scan of the electric and magnetic sector field strengths of the second mass spectrometer was used to transmit the fragment ions arising from collisions with a stainless steel surface. The resulting mass spectra contained broad metastable ion peaks produced by the dissociation of MH⁺ and [M + Cat]⁺ ions before the second magnetic sector, in the fourth field-free region of the instrument.     

Examination of complex oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry on time-of-flight and magnetic sector instruments

Matrix-assisted laser desorption/ionization (MALDI) spectra of underivatized oligosaccharides of the type attached to asparagine in glycoproteins (N-linked oligosaccharides) were examined with linear time-of-flight (TOF) and magnetic sector instruments using 2,5-dihydroxybenzoic acid (2,5-DHB), α-cyano-4-hydroxycinnamic acid, sinapinic acid, 1,4-dihydroxynaphthalene-2-carboxylic acid or 2-(4-hydroxyphenylazo)benzoic acid (HABA) as the matrices. All compounds formed abundant [M + Na]⁺ ions with the strongest signals being obtained from 2,5-DHB after recrystallization of the initially dried sample spot from ethanol. Only traces of fragmentation were detected from neutral oligosaccharides on the TOF system but more abundant fragment ions (about 5% relative abundance) were present in the spectra from the magnetic sector instrument. Fragmentation was dominated by Y-type glycosidic cleavages (Domon and Costello nomenclature) between all sugar residues yielding sequence and branching information. Sialic acid-containing oligosaccharides generally produced the sodium adduct of the sodium salt and gave much weaker signals than the neutral sugars in the positive-ion mode. There was also considerable loss of the sialic acid moleties as the result of fragmentation on the magnetic sector instrument. The least fragmentation of both neutral and acidic sugars was caused by 2.5 DHB, which proved to be the most appropriate matrix for examination of oligosaccharide mixtures. Much better resolution of the oligosaccharides was obtained than by traditional methods such as the use of Bio-Gel P-4 gel filtration column chromatography. It is worth noting also that the measurements were considerably faster (a few minutes as opposed to about 16 h). In addition, no radiolabelling was necessary as required for detection on the P-4 columns. Mixtures of oligosaccharides from several glycoproteins (ribonuclease B, human immunoglobulin G (IgG) transferrin, bovine fetuin and chicken ovalbumin) were examined and the patterns of the identified oligosaccharides were found to agree closely with the known compositions of the sugar mixtures. The mass spectrometric resolution on the magnetic sector instrument was very much better (up to 3000, FWHM) than could be obtained with the linear TOF systems (200–400). The technique was used as a detection system for the products of exoglycosidase digestion in experiments to determine the detailed structure of the oligosaccharide chains from human IgG.     

Design and performance of a plasma-source mass spectrograph

A plasma-source, simultaneously detecting, Mattauch-Herzog mass spectrograph for multi-element analysis was developed. Simultaneous detection should improve throughput, precision, and sensitivity over those instruments that use quadrupole or sector mass spectrometers. The new instrument is compact (approximately 80 cm in length) and is designed to detect a complete atomic mass spectrum in two mass windows that straddle but avoid argon. The design and some figures of merit are presented. With a dc glow discharge source, a precision of 0. 5% determined from eight consecutive 10-s images was obtained for Cu isotope ratios in Naval Brass B. With the same sample, nickel and lead were detected at limits of 8 and 1 ppm. Measured Mg isotope ratios with an inductively coupled plasma source were within 4% of the expected values. The resolution at full width at half maximum is currently limited to approximately 60, in part because of poor peak shape. The origin of this peak shape has been determined to lie within the array detector and possibly results from its interaction with the fringing magnetic fields produced by the second sector.     

Comparative performance study of ICP mass spectrometers by means of U “isotopic measurements”

The performance of four commercially available ICPMS instruments of three different types was compared by means of uranium “isotopic measurements”. Examined were two quadrupole sector (different generation, different manufacturer), one single detector double focusing magnetic sector and one multiple collector double focusing magnetic sector instruments. The same samples of the IRMM-072 series were used under routine conditions to measure the ²³³U/²³⁵U and the ²³³U/²³⁸U ratios which, in these samples, vary over almost three orders of magnitude from ～ 1 to ～ 2 · 10^–3. Within expanded (k = 2) uncertainties, good agreement was observed between the certified values and the data internally corrected for mass-discrimination effects. The magnitude of the evaluated uncertainties was different for each type of instrument. With the multiple collector instrument, expanded uncertainties varied from ± 0.04% to± 0.24% for the ²³³U/²³⁵U ratio, and from ± 0.08% to ± 0.27% for the ²³³U/²³⁸U ratio. They were ～ 1 to 5 times larger with the single detector magnetic sector instrument, and ～ 10 to 25 times larger with both quadrupole sector instruments. With the multiple collector instrument, repeatability of the measurements seemed to be limited by the difficulty of correcting properly for instrumental background, whereas with the single detector magnetic sector instrument the counting statistics was the only limitation (on smallest ratios). Apparent mass-discrimination was clearly found to be larger but more reproducible (and hence easier to correct for) in the case of magnetic sector instruments than for both quadrupole sector instruments. If space charge effects were the main source of mass-discrimination for all instruments, these results are in contradiction with the hypothesis of the size of mass-discrimination decreasing with the acceleration voltage. With the single detector magnetic sector instrument in particular (when operated by changing the ion energy only), our results pointed at more than only one major source of mass-discrimination, with variable size depending on the ratios measured.     

Preparative separation of a multicomponent peptide mixture by mass spectrometry

We report on the first multiplex preparative separation by mass spectrometry of bio-organic molecules in the 200-350 Da mass range that is typical for synthetic drugs. A five-component mixture consisting of two di- and three tripeptides has been separated by mass using a specially designed mass spectrometer. The instrument for preparative separations consists of an electrospray ionization (ESI) source, ion transfer optics, an electrostatic sector, and an inhomogeneous-field magnetic mass analyzer that achieves linear mass dispersion of ion beams. Protonated peptides produced by electrospray were separated, nondestructively landed on a 16-channel array of dry collector plates, and reconstituted in solution. The preparation procedures and the instrumental conditions have been optimized to maximize the ion currents. The significant features of the special mass spectrometer are high ion currents and simultaneous separation and collection of mixture components.     

Tandem mass spectrometric accurate mass performance of time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry: a case study with pyridine derivatives

The interpretation of mass spectra is a key process during compound identification, and the combination of tandem mass spectrometry (MS/MS) with high-accuracy mass measurements may deliver crucial information on the identity of a compound. Obtaining accurate mass data of fragment ions in MS/MS reveals the particular problem of mass calibration when a lockmass, which is frequently used to obtain accurate masses in MS, is absent. An alternative technique is to recalibrate the MS/MS spectrum using a reference MS/MS spectrum acquired under the same conditions. We have tested and validated this approach using a hybrid quadrupole/orthogonal acceleration reflectron-type time-of-flight (TOF) mass spectrometer. The results were compared with those obtained under similar conditions on a Fourier transform ion cyclotron resonance (FT-ICR) instrument. We found that the mass accuracy observed with such an "external" recalibration on the TOF instrument in MS/MS is identical to what can be obtained on a similar instrument operating in one-dimensional MS mode using the lockmass technique. However, mass accuracy in both cases is one order of magnitude inferior to that obtained using FTMS, and also inferior to that observed using sector field MS when operated at comparable resolution. Nevertheless, for small (<200 Da) molecules, this mass accuracy was still sufficient to have the "true" elemental composition identified as the first hit in about 70% of all cases. It was possible to elucidate the fragmentation mechanism of eight azaheterocycles containing a pyridine moiety, where the accurate mass data from the TOF instrument allowed distinction between two alternative fragmentation pathways.     

Gas-phase ion chemistry and organic chemistry-the story of a hybrid six sector mass spectrometer--the "AutoSpec 6F"

The AutoSpec 6F mass spectrometer is a large, floor standing instrument comprising a pair of commercial EBE geometry (AutoSpec) mass spectrometers coupled in series to provide an hybrid EBE-EBE configuration, (E and B being respectively electrostatic and magnetic sectors.) It was designed in close collaboration between Professor R. Flammang and VG Analytical in Manchester, UK. It was equipped with five collision cells and allowed the recording of high energy CID (collision induced dissociation), MIKES (mass analyzed ion kinetic energy spectrometry) and NRMS (neutralization re-ionization mass spectrometry) data as well as consecutive MSn analyses. The field-free regions between sectors allowed the study of unimolecular decomposition products from long-lived metastable ions. The mass spectrometer became even more versatile when an RF-only quadrupole collision cell was installed between the second and the third electric sector. This allowed the study of associative ion/molecule reactions in the low kinetic energy regime. Bimolecular chemical reactions were performed inside the quadrupole cell when a neutral reagent was introduced and the reaction products were analyzed by high energy CID in the downstream sectors. This paper tells the history and summarizes the capabilities of this versatile instrument.     

Detection of femtomole and sub-femtomole levels of peptides by tandem magnetic sector/reflectron time-of-flight mass spectrometry and matrix-assisted laser desorption ionization

This article describes results of low-level (sub-femtomole) detection of peptides by matrix-assisted laser desorption ionization. The matrix-assisted laser desorption ionization method can be used for low-level detection of the parent ion, either [M + H]⁺ or [M + Na]⁺, and collision-induced dissociation of the parent ion can be performed at the picomole level. The instrument used for these studies is a novel high-performance magnetic sector (electric(E)/magnetic(B) sector)/reflectron time-of-flight (TOP) tandem mass spectrometer (EB/TOF).     

Primary to quaternary protein structure determination with electrospray ionization and magnetic sector mass spectrometry

Electrospray ionization with a forward-geometry magnetic sector mass spectrometer was used for collisionally activated dissociation studies of multiply charged polypeptides and for studying non-covalently bound protein systems. The high-resolution capabilities of a high-performance instrument allow the resolution of isotopic contributions for product ions and molecular ion species. Determination of product ion charge states by this method reduces difficulties in the interpretation of product ion mass spectra from multiply charged precursors, which are generated either in the atmospheric pressure/vacuum electrospray interface or in the collision chamber of the mass spectrometer. Extended tandem mass spectrometric experiments have the potential for sequencing larger polypeptides. However, evidence for isomerization of gas-phase product ions from substance P and substance P analogues was observed, complicating the interpretation of product ion spectra. Non-covalent complexes can also be studied by electrospray ionization magnetic sector MS. The higher m/z range of such an instrument is a major advantage for studying weakly bound systems, such as heme–protein systems (myoglobin, hemoglobin) and protein aggregates (concanavalin A), because of their tendency to form complex ions with relatively low charge states.     

Sequencing of new beauverolides by high-performance liquid chromatography and mass spectrometry.

Mass spectrometry (MS) and tandem mass spectrometry (MS(n)) were used for the identification of beauverolides in the fermentation broth of Beauveria bassiana and for evaluation of the purified fraction obtained by sublimation of beauverolides. Besides being a new efficient route for purification of beauverolides, sublimation provided an enrichment of new minor lipophilic beauverolides of lower molecular weight from the original complex mycelial extract. The product ion collision-induced dissociation (CID) spectra obtained on an ion trap (electrospray ionization), the in-source CID mass spectra on a sector instrument (atmospheric-pressure chemical ionization) and the post-source decay matrix-assisted laser desorption/ionization mass spectra of beauverolides were compared and evaluated. All MS(n) experiments started with singly charged precursor ions. The following two new representatives of this group of compounds were identified by high-performance liquid chromatography and MS (HPLC/MS): cyclo-(3-hydroxy-4-methyloctanoyl-valyl-alanyl-leucyl) and cyclo-(3-hydroxy-4-methyloctanoyl-tyrosyl-alanyl-leucyl). Individual structures were confirmed by preparative isolation and nuclear magnetic resonance spectroscopy. The structure of a third novel and minor beauverolide was tentatively assigned by HPLC/MS only as cyclo-(3-hydroxy-4-methyldecanoyl-valyl-alanyl-Lxx), Lxx = leucyl, isoleucyl, or allo-isoleucyl.     

Capabilities of double focussing magnetic sector-ICP-MS for the determination of trace elements in body fluids (blood, blood serum, urine) at the example of control materials

The capability of double focussing magnetic sector inductively coupled plasma mass spectrometry (HR-ICP-MS) for the determination of trace elements in different body fluids was investigated in commercially available control materials. The elements included in this study were: Al, As, Cd, Cr, Co, Cu, Hg, Mn, Ni, Pb, Sb, Se, Tl and Zn. Sample preparation, instrument stability and the results, which are in very good agreement to the given values, are presented and discussed. Received: 27 August 1997 / Revised: 19 November 1997 / Accepted: 23 November 1997     

Capabilities of double focussing magnetic sector-ICP-MS for the determination of trace elements in body fluids (blood, blood serum, urine) at the example of control materials

The capability of double focussing magnetic sector inductively coupled plasma mass spectrometry (HR-ICP-MS) for the determination of trace elements in different body fluids was investigated in commercially available control materials. The elements included in this study were: Al, As, Cd, Cr, Co, Cu, Hg, Mn, Ni, Pb, Sb, Se, Tl and Zn. Sample preparation, instrument stability and the results, which are in very good agreement to the given values, are presented and discussed. Received: 27 August 1997 / Revised: 19 November 1997 / Accepted: 23 November 1997